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Nephropathic cystinosis: 10th anniversary of the CTNS gene Elena Levtchenko, MD, PhD University Hospital Leuven, Belgium Definitions • Cystinosis – an autosomal recessive disease caused by lysosomal accumulation of cystine due to defective exodus of cystine out of the lysosomes • Cystinosis – incidence ~1:200,000 – clustering in some populations • Cystinosis – most common cause of inherited generalized proximal tubular dysfunction (renal Fanconi syndrome) Clinical forms • Infantile form: – Fanconi syndrome ~ 3-6 months – end stage renal disease (ESRD) ~ 10 years • “Late-onset” (juvenile) form: – later onset (often during puberty) – mild tubulopathy, more pronounced proteinuria, (even in nephrotic range) – later progression to ESRD • Ocular form • Overlap between ocular and juvenile forms (Servais et al. 2008) 3 Extra-renal involvement Eye – photophobia – kerathopathy – retinal blindness Endocrine organs – hypothyroidism – diabetes mellitus – male hypogonadism Neuromuscular disease – myopathy Neurological complaints – epilepsy – mental deterioration – cerebellar and pyramidal signs – stroke-like episodes Liver disease, exocrine pancreas deficiency 4 Gahl et al. 2002 Treatment with cysteamine cystinosin cytoplasm cytoplasm cystine NH2 cysteamine cysteine – cysteamine cysteine COOH CH lysosome CH CH2 CH2 CH2 CH2 CH HS S + CH2 NH2 NH2 CH COOH COOH SH CH2 S NH2 lysosome NH2 cystine COOH + S S CH2 CH2 NH2 cysteine transporter 5 CAT- transporter Efficiency of cysteamine in cystinosis • Cysteamine postpones the deterioration of the renal function (Markello et al. 1993) • Cysteamine protects extra-renal organs and should be administered also after renal transplantation (Gahl et al. 2002) • Topical cysteamine drops (0.5%) dissolve corneal cystine crystals (Tsilou et al. 2002). New viscous cysteamine eye preparations (Bozdag et al. 2008) BUT: • Cysteamine does not reverse renal Fanconi syndrome • Many patients still develop ESRD and extra-renal complications (at later age) • Major compliance problems due to: - dose regimen every 6 hours (daily dose 1.3-1.95 g/m2) - gastro-intestinal complaints (Dohil et al. 2006) - bad breath and sweat odor due to formation of dimethylsulfide (Besouw et al. 2007) cystinosis heterozygotes controls Cystine dysposal from granular fraction of normal, heterozygous and cystinotic cells loaded with [35S] cystine dimethyl ester CTNS gene structure (17p13, 23 kb) TAG ATG 1 2 3 c 4 4 5 6 7 8 9 10 11 12 Cystinosin: predicted structure YFPQA GYDQL Chergui et al. 2001 9 Most common mutation in European population: 57 kb deletion: CTNS gene, CARKL gene (Town et al. 1998, Wamelink et al. 2008) Functional assay of cystinosin [35S] - C-S-S-C + H+ N C C cytoplasm lysosome N Kalatzis et al. 2001, 2004 Expression of Cystinosin-LKG is not restricted to lysosomes Taranta et al. 2008 Pathogenesis of cystinosis • How cystinosin defects lead to cell dysfunction in cystinosis is still unknown • Existing hypotheses from in vitro studies: – Cystinosin dysfunction leads to defective gluthation synthesis in cystinosis enhanced oxidative stress leading to cell damage (Levtchenko et al. 2005, Mannucci et al. 2006, Laube et al. 2006) – Increased sensitivity of cystinotic cells to apoptotic stimuli due to cysteinylation of protein kinase C (Park et al. 2002, 2006) enhanced cystinotic cell death – ATP deficiency cell dysfunction and Fanconi syndrome al. 1991, Ben-Nun et al. 1993, Foreman et al. 1995, Wilmer et al. 2006) – No prove in humans or in ctns -/- mice model (Coor et interstitium lumen nucleus cytosole lysosome - LKG influence on gene expression cystine cysteine oxidative redox state - LKG -glut-cys GSSG cystinosin cystine reductase GCS GSSG/GSH Cysteinylation of PKC- cystine cystine cystine GS H2O2 GSH oxoglutarate carrier dicarboxilate carrier glucose GSH reductase mitochonrion GSH GSSG glycolytic pathway GSH GSH peroxidase Stimulation of apoptosis . O2- 14 SOD O2-. ADP eI H+ lipid peroxides H2O2 H2O2 II III H+ SOD IV O2-. . O2- ATP + P V e- citric acid cycle pyruvate NADH FADH2 pyruvate carrier H+ Disturbed ATP metabolism Possible functions of cystinosin-LKG • Cystinosin-LKG has probably other functions in the cell additionally to lysosomal cystine transport: – Cystine-binding in the cytosole: regulate cystine - cysteine pool in the cell and prevent the formation of disulfide compounds – Alternative plasma membrane cystine transporter Molecular therapy, vol.16, No.8, 1372-1381, aug.2008 Liver from 3-month old ctns -/- mice Obstacles of gene therapy in cystinosis: • Gene should be delivered to all organs • Use of viral vectors Liver from 6-month old ctns -/- mice • Immune response • Short duration of cystine-lowering effect • Gene transfer should be administered at early age Summary • Discovery of cystinosin in 1998 opened a new chapter of diseases caused by defects of lysosomal transport proteins. • New data indicate that the expression of cystinosin is not restricted to the lysosomal membrane. Ongoing studies will identify new functions and regulators of cystinosin activity new insights into the pathogenesis of cystinosis. • Gene therapy in cystinosis is feasible and is a subject of intensive research. Many obstacles should be overcome. Thanks to Cystinosis research group Nijmegen/Leuven Bambino Gesù Children hospital, Rome F. Emma A. Taranta VUMC, Amsterdam L.Monnens M. Wilmer M. Besouw B. Van den Heuvel H. Blom M. Wamelink QUESTIONS? Lysosomal membrane transport H+ ATP ADP lysosome Acid hydrolases: Proteases Glycosidases Nucleases Phosphatases Sulphatases H+ ? H+ Substrates: Amino acids Monosaccharides Nucleosides Di/tripeptides Inorganic ions Vitamins New insights into the lysosomal membrane transport • Competition experiments in isolated lysosomes predicted ~20 lysosomal transporters (Pisoni, Thoene 1991) • Analogous to cystinosin: functional assays of lysosomal membrane proteins (genotype-phenotype correlations in sialic acid storage disease (Morin et al. 2004) • Proteomics studies of lysosomal membrane (Schroder et al. 2007) – Identification of new transporters (glucose (GLUT8), myo-inisitol (HMIT), Zn transporter, KCC1…) • New genes responsible for lysosomal transport disorders (mucolipidosis type IV (MCOLN1), infantile malignant osteopetrosis (ClC7), late infantile neuronal ceroid lipofiscinosis (CLN7) corneal 22 cystine crystals cystinotic kerathopathy cystinotic retinopathy nucleus cystine cystine cysteine H+ cystinosin cystine cysteine H+ cystine cysteine lysosome protein degradation cytoplasma 23 n= 67 n = 32 n = 17 24 Markello et al. 1993 Cystine accumulation in cystinosis Kidney: 200 - 400 x normal Liver: 80 - 1000 x normal Muscle: 40 - 70 x normal Brain: 5 - 20 x normal Stokes et al. 2008 Wilmer et al. 2008